Optimization of non-ATP competitive CDK/cyclin groove inhibitors through REPLACE-mediated fragment assembly.

A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way, and this was achieved for compounds targeting the cyclin-dependent kinase 2 (CDK2) substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore, the structure-activity relationship of a bis(aryl) ether C-terminal capping group mimicking dipeptide interactions was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds.

Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements.

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove, which to date has not been carried out in a systematic fashion. Collectively, the data presented provide new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and antitumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.

Non-ATP competitive protein kinase inhibitors as anti-tumor therapeutics.

Alternative approaches for inhibitor development in targeting sites other than the ATP cleft are increasingly being pursued in the search for new therapeutics based on inhibition of protein kinases. While recently approved kinase inhibitor drugs offer benefit in cancer treatment, further advances are required to affect tumor selective cell killing, avoid off-target related toxicities and improve survival rates. Protein-protein interactions involved in kinase regulation and substrate recognition as well as exploiting allosteric pockets, offer the potential for selectivity and avoid decreased efficacy as a result of competition with high intracellular ATP concentrations. We discuss several preliminary examples where regulatory and substrate binding sites present potential druggable interfaces. These include the cell cycle targets which are the cyclin-dependent and polo-like kinases among several others.